Rodent studies have demonstrated critical roles of regulatory T cells (Treg) in transplant tolerance, raising expectations that adoptive Treg therapy may eliminate the dependence of transplant patients on CNI-based immunosuppression (IS),- a major cause of morbidity and mortality. Recent use of alternative therapy, in particular the costimulation blocking (CoSB) agent belatacept, while improving long-term renal graft function, has led to increased rates of rejection. While T cell depletion (ATG) at the time of transplant reduces dependence on CNI, homeostatic T cell proliferation results in preferential expansion of memory T cells (Tmem),- a major barrier to tolerance induction. Combined T cell depletion plus belatacept can prevent rejection, but tolerance is not achieved. While clinical trials are underway to evaluate Treg in organ transplantation, their functional plasticity and potential conversion to pathogenic T cells raise valid concerns, Better understanding of the mechanisms that may alter Treg function after their infusion in graft recipients is essential for safe and effective implementation of Treg therapy. We have shown recently that early polyclonal Treg infusion into lymphodepleted NHP is associated with enhanced graft injury and Tmem recovery. IL-2 and transforming growth factor beta 1 (TGF?1) are critical for Treg development and low dose IL-2 selectively expands endogenous Treg in NHP and humans. Also, TGF?1 signaling is essential for prevention of inflammatory responses in lymphopenic hosts. In rodents, infusion of huTGF?1/Fc in combination with rapamycin enhances graft survival. In collaboration with the NIAID NHP Reagent Resource we have generated an agonistic TGF?1/Fc protein for use in NHP. Our preliminary data show that this TGF?1/Fc reduces rhesus T cell proliferation synergistically with rapamycin and promotes Treg in the presence of IL-2. Circulating TGF?1/Fc can be detected for several days after its infusion into lymphodepleted rhesus. Based on these findings, we propose to evaluate, the influence of donor alloractive- reactive Treg (arTreg) therapy in combination with IL-2 and TGF?1/Fc on renal graft survival in the rhesus model. All three Specific Aims will be accompanied by mechanistic and biomarker studies and be highly interactive with Project 1 and Cores A and B. Aim 1: To determine the influence of ex-vivo expanded autologous arTreg on renal allograft survival in rhesus macaques given ATG, belatacept and rapamycin (ABR) IS, and underlying mechanisms Aim 2: To assess whether low-dose IL-2 can enhance the suppressive function and numbers of ex- vivo expanded arTreg in graft recipients given ABR IS and further improve graft survival Aim 3: To evaluate the influence of co-administered TGF?1/Fc and IL-2 on the stability and suppressive function of ex-vivo expanded arTreg in graft recipients given ABR IS and promote operational tolerance